Single or multiple brief periods of myocardial ischemia prior to a prolonged period of ischemia have been shown to produce a marked reduction in myocardial infarct size, a phenomenon termed ischemic preconditioning (IPC). IPC occurs in two phases, an acute phase in which the cardioprotective effects only persist for 1-2 hours and a delayed phase which reoccurs at 12-24 hours and persists for 48-96 hours. Our laboratory was the first demonstrate that endogenous opioids acting via a delta (delta) opioid receptor and enhanced opening of the adenosine-triphosphate sensitive potassium channel (KATP channel) were key factors in triggering and maintaining acute and delayed IPC in the intact rat heart. Based on these findings, the long- term goal of the present proposal is to elucidate intracellular signalling pathways by which delta1-opioid receptor activation leads to acute and/or delayed PC in the rat heart. The major hypothesis to be tested is that activation of the delta1-opioid receptor produces acute and delayed PC by activating several intracellular kinase pathways consisting of tyrosine kinase (TK), protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) cascade which leads to activation of the mitochondrial KATP channel (mito KATP) and cardioprotection. Experiments will be performed in isolated buffer-perfused rat hearts and intact blood-perfused rat hearts. By use of selective pharmacological probes, immunocytochemistry and Western blot analysis, we will determine the role of TK, different isoforms of PKC and the 3 major components of the MAPK pathway, extracellular signal- regulated kinase (ERK 1/2), Jun N-terminal kinase (JNK) and p38 MAPK in opioid-induced PC. We will use 2 indices, infarct size at the organ level and mitochondrial function at the subcellular level, to assess myocardial injury following prolonged ischemia and reperfusion in isolated and intact hearts. Mitochondrial KATP channel function will be assessed by measurements of ATP synthesis rates, mitochondrial membrane potential and mitochondrial oxygen consumption. Mitochondria will be harvested from control, opioid- and IPC-treated hearts in the presence and absence of agonists or antagonists of mito KATP to determine the role of this subcellular organelle in cardioprotection. Mechanisms of delayed PC produced by opioids will be compared to the heat shock response as a standard of comparison. The results of the proposal are novel and have great clinical significance and may result in an effective new means of treating patients with acute or chronic ischemic heart disease. This project is particularly exciting since many opioid agonists are already available to physicians and a long period of drug development may not be necessary before implementing this concept in patients.